CD4+CD25+ regulatory T cells (TRegs) are critical for the acquisition of peripheral allograft tolerance. However, it is unclear whether TRegs are capable of mediating alloantigen-specific suppressive effects and, hence, contributing to the specificity of the tolerant state. In the current report we have used the ABM TCR transgenic (Tg) system, a C57BL/6-derived strain in which CD4+ T cells directly recognize the allogeneic MHC-II molecule I-Abm12, to assess the capacity of T Regs to mediate allospecific effects. In these mice, 5-6% of Tg CD4+ T cells exhibit conventional markers of the TReg phenotype. ABM TRegs are more effective than wild-type polyclonal TRegs at suppressing effector immune responses directed against I-Abm12 alloantigen both in vitro and in vivo. In contrast, they are incapable of suppressing responses directed against third-party alloantigens unless these are expressed in the same allograft as I-Abm12. Taken together, our results indicate that in transplantation, TReg function is dependent on TCR stimulation, providing definitive evidence for their specificity in the regulation of alloimmune responses.